To further substantiate our earlier claim that unicellular organisms make vertebrate peptide hormones, we have extended our studies. Thus, we studied additional strains of tetrahymena pyriformis, a citiated protozoan as well as three new strains of E. coli. In each instance, we have found material very similar to insulin. These similarities include chromatography on ion exchange sephadex columns, reverse phase hydrophobic interactions an radioimmunoassay as well as bioassay. The prouction of insulin by both tetrahymena and E. coli during their growth cycles were studied; insulin can be recovered from extracts of the cells as well as from the media during the logarithmic growth phase. In a preliminary fashion in extracts of tetrahymena, other hormones are found to be present: cholecystokinin, glucagon, arginine, vasotocin, and relaxin. Further extension of this study shows that insulin is present in the chick embryo before the beta cell is recognizable which extends observations that insulin is native to organisms that lack pancreatic islets, including flies, worms, and microbes.